QC and filter seurat object

Parameter settings

Import Seurat object

Add filter meta data to Seurat object

Filter cells by rank plot

Empty droplets were determined with CellRanger V3.0.2 Lun et al., 2019 EmptyDrop heuristic. RNAse activity of granulocytes might be wrongly identified as empty cells by CellRanger.

Typical Sample A steep drop-off is indicative of good separation between the cell-associated barcodes and the barcodes associated with empty GEMs. A ideal barcode rank plot has a distincitve shape, which is referred to as a "cliff and knee".
Heterogeneous Sample Heterogeneous populations of cells in a sample result in two "cliff and knee" distributions. However, there should still be clear separation between the bacodes.
Compromised Sample Round curve and lack of steep cliff may indicate low sample quality or loss of single-cell behavior. This can be due to a wetting failure, premature cell lysis, or low cell viability.
Compromised Sample Defined cliff and knee, but the total number of barcodes detected may be lower than expected. This can be caused by a sample clog or inaccurate cell count.

Filter by CellRanger class

Filter by UMI and Feature count

The Progenitor NaCl sample show a high MT%. It would be interessting to see whether those are apoptoitic cells or if they have a increased energy demand. That could be done by correlating the MT% with complementary nuclear mitochondrial gene expression from the OXPHOS system.

Correlate percent Mt with OXPHOS nDNA model

Frederick et al., 2020 https://www.nature.com/articles/s41598-020-63448-z

Box plots

SingleR annotation

SingleR identifies marker genes from the reference and uses them to compute assignment scores (based on the Spearman correlation across markers) for each cell in the test dataset against each label in the reference. The label with the highest score is the assigned to the test cell, possibly with further fine-tuning to resolve closely related labels.

first.labels: Labels before fine-tuning
labels: Labels after fine-tuning
pruning: labels after pruning

Ery score

Compute module scores

Histocompatibility Class I

H2-K1 - Class I histocompatibility antigen, kappa-B alpha chain
H2-D1 - Class I histocompatibility antigen, D-B alpha chain
H2-L1 - not found

Histocompatibility Class II

H2-Aa - Class II histocompatibility antigen, A-B alpha chain
H2-Ab1 - Class II histocompatibility antigen, A beta chain

H2-Eb1 - Class II histocompatibility antigen, I-E beta chain
H2-Eb2 - Class II histocompatibility antigen, E beta chain

Info: The antigens presented by class I peptides are derived from cytosolic proteins while calss II derived from extracellular proteins.

Surface marker

Cd4 - T-cell surface glycoprotein CD4, Co-receptor for MHC class II, active on T-helper cells and triggers differentiation of monocytes into functional mature macrophages.
Cd8 - T-cell surface glycoprotein CD8, Co-receptor for MHC class I, CTL activattion and thymic selection of Cd8+ T cells.

T-cell TCR-CD3 complex

Trac - T cell receptor alpha constant
Trbc1 - T cell receptor beta constant 1
Trbc2 - T cell receptor beta constant 2
Trdc - T cell receptor delta constant
Trgc1 - T cell receptor gamma constant 1
Trgc2 - T cell receptor gamma constant 2

Cd247 - T-cell surface glycoprotein Cd3 zeta chain (Cd3z)
Cd3g - T-cell surface glycoprotein Cd3 gamma chain
Cd3e - T-cell surface glycoprotein Cd3 epsilon chain
Cd3d - T-cell surface glycoprotein Cd3 delta chain

B-cell Immonogloblin complex

Naive B-cells produce the following Ig classes:
Ighm - Immunoglobulin heavy constant mu (naive B-cells)
Ighd - Immunoglobulin heavy constant delta (naive B-cells)

Through isotope switching the following Ig classes can be produced:
Ighg1 - Immunoglobulin heavy constant gamma (Mouse with Igh1b have Igg2c isotope instead of Igg2a)
Ighg2a - Immunoglobulin heavy constant gamma (NA)
Ighg2b - Immunoglobulin heavy constant gamma
Ighg2c - Immunoglobulin heavy constant gamma
Ighg3 - Immunoglobulin heavy constant gamma
Igha - Immunoglobulin heavy constant alpha
Ighe - Immunoglobulin heavy constant epsilon (NA)

Igkc - Immunoglobulin kappa constant (light chain)
Iglc - Immunoglobulin lambda constant (light chain)

Compute cell cycle score

SCTransform and dimensional reduction (sample_group)

Identify outlier cluster

Sample replicates

Myeloid CpG

Progenitor CpG

Myeloid NaCl

Progenitor NaCl

Remove cluster from so_qc (sample group)

SCTransform and dimensional reduction (treatment)

SCTransform + Cell cycle Regression and dimensional reduction (treatment)

Save output

Session info